The invention relates to methods for monitoring immunological function in a human or animal. More particularly, the present invention relates to accurately measuring the production of cytokines in vivo.
There have been many attempts to measure endogenous cytokines in blood and other body fluids. However, in reviewing these reports, it is apparent that there is wide variation in the reported results with regard to cytokine concentration in the blood and to fluctuations of cytokine concentration in the blood. Measurement of cytokine production in vivo is an important part of many experimental and therapeutic studies. Interpretation of results has been difficult, however, for several reasons:
1. Studies that examine cytokine secretion ex vivo are relatively easy to perform, but may not reflect cytokine production in vivo.
2. Cytokine levels in serum or other biological fluids can be measured in some cases; however, levels are often negligible because many cytokines have a very short in vivo half-life. In addition, increased absorption of cytokines by receptors on activated cells may accelerate catabolism of cytokines, so that serum levels will not correlate directly with secretion.
3. Cytokine mRNA levels can be measured directly after removal of cells or organs, and presumably, accurately reflect in vivo cytokine mRNA levels; however, protein production and secretion doesn""t always vary directly with mRNA levels.
4. Cytokine-containing cells have been identified by staining procedures; however, the small number of such cells in the absence of in vitro restimulation and subjectivity in identifying such cells by staining techniques place limits on the usefulness of these techniques. In addition, it is not known if all cytokine-secreting cells have identical levels of stored, intracellular cytokine, so that intensity of staining and rate of secretion do not necessarily correlate directly.
5. ELISPOT assays for cytokine-secreting cells can be performed on cells right after they are removed from an animal; however, enumeration of cytokine-secreting cells with this technique is somewhat subjective, and this technique may detect only those cells that secrete the largest amount of cytokine, may favor selective detection of those cells that contain stores of a preformed cytokine, and, even if the assay involves only a short incubation period, may not reflect in vivo cytokine production because of the disruption of normal architecture, the possibility that some activated cells may be difficult to remove intact from organs, the possibility that the trauma of preparing single cell suspensions may modify cytokine secretion, etc.
Many reports indicate that cytokines (i.e., IL-2) are not detectable in normal subjects using immunoassays. Cytokines are difficult to measure in serum for several reasons: 1) rapid renal excretion; 2) catabolism; 3) cellular utilization; and 4) binding to molecules, such as soluble receptors that mask the cytokine active site.
It would be of great benefit if one could easily, accurately and reproducibly measure in vivo cytokine production by sampling body fluids, such as blood. This would create a useful window not only into the immune system but into a myriad of physiologically interacting processes. Such a tool would be useful in a variety of settings, allowing the collection of data of importance to basic medical sciences, clinical medicine, epidemiology and the forensic sciences.
U.S. Pat. No. 4,486,530, David et aL, issued Dec. 4, 1984, discloses a xe2x80x9cTwo-sitexe2x80x9d or xe2x80x9csandwichxe2x80x9d immunometric assay technique for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies. These are described and compared to conventional assays using polyclonal antibodies. Also described are inhibition assays using complexes of antigens with a monoclonal antibody. This reference discloses methods for detecting and/or determining the concentration of antigenic substances in fluids such as serum.
U.S. Pat. No. 5,587,294, Tamarkin et al., issued Dec. 24, 1996, discloses methods for measuring endogenous cytokines in blood. The method accurately measures the cytokines in the blood in the presence of substances that bind the cytokines thereby causing the measurement of the cytokines by conventional methods to give inaccurate results. The Tamarkin et aL patent also describes the non-invasive measurement of cytokines in biological fluids such as saliva and nasal secretions. Finally, the procedure described in Tamarkin et al., allows one to monitor the level of cytokines in the blood during treatment of a human or animal with cytokines.
Others have shown the prolongation of in vivo effects of exogenous cytokines by injection of cytokine--anti-cytokine antibody complexes. (J. ImmunoL 151:1235, 1993). This reference describes a technique whereby animals are injected with preformed complexes of a cytokine and an anticytokine MAb. The antibody acts as a carrier protein for the cytokine, slowly dissociating and thereby increasing the period of time during which active cytokine is present in an animal. This paper does not mention the use of this technique for determining cytokine production in vivo. It does reference other publications that mention that endogenously produced anti-cytokine antibodies may enhance the half-life and activity of endogenously produced cytokines in vivo; however, these papers also did not mention this as a possible technique for measuring endogenous cytokine production. The reference does not disclose the idea of labeling an injected anticytokine antibody to facilitate detection of cytokine/anti-cytokine antibody complex.
U.S. Pat. No. 5,612,034, Pouletty et al., issued Mar. 18, 1997, provides first and second compounds, where the first compound is administered to a mammalian host into blood for covalent bonding to blood components, where the components have an extended lifetime in the blood stream. The first compound comprises an active functionality and an agent of interest or a first binding entity. A second compound may be subsequently administered to the patient, which comprises a second binding entity, complementary to the first binding entity and an agent of interest. By virtue of binding to long-lived blood components, the half-life of the agent of interest is greatly extended in vivo.
It would be of great benefit if one could easily, accurately and reproducibly measure the concentrations of various endogenous cytokines in blood in vivo. This would provide information not only of the immune system but of a variety of physiologically interacting processes. Such tools would be useful in a variety of settings, allowing the collection of data of importance to basic medical sciences, clinical medicine, epidemiology and the forensic sciences.
What is needed is a reliable method of measuring endogenous cytokine production by sampling blood, which is only minimally affected by cytokine catabolism, utilization, excretion, or binding to endogenous cytokine binding proteins.
The present invention provides methods for measuring the endogenous level of a target analyte which may be a hormone, drug or other analyte in a human or animal. The target analyte is preferably a macromolecule, more preferably a protein, and most preferably a cytokine.
The present invention is an immunoassay for use in detecting and monitoring endogenous cytokine production. Prior methods have been unable to accurately measure cytokine production because of rapid excretion, catabolism, and utilization of cytokines as well as the binding of cytokines to endogenous cytokine binding proteins, which can interfere with detection. The present invention obviates all of these difficulties.
The present invention provides the capability of measuring xe2x80x9cbasal,xe2x80x9d as well as xe2x80x9cstimulated,xe2x80x9d cytokine production. The present invention provides a new tool for monitoring these chemical communication signals and their dysregulation in the face of challenges by pathogens, chemicals, therapeutics as well as by biobehavioral factors.
The present invention is especially useful in measuring proteins secreted into extracellular fluid, including blood, and includes, but is not limited to, proteins from the group consisting of interleukins 1 through 18, interferon-alpha, interferon-beta, interferon-gamma, lymphotoxin, and tumor necrosis factor-alpha. It is contemplated that the present invention will be useful in detecting and quantifying other cytokine-like molecules in the blood that have not yet been characterized.
In general, the present invention provides a method of measuring the production of a target analyte of interest in a human or animal, comprising the steps of:
a. injecting a human or animal with an appropriate amount of targeting moiety capable of binding specifically to the target analyte;
b. allowing the targeting moiety to circulate through the injected human or animal for a time sufficient to bind to the target analyte of interest and form a targeting moiety: target analyte conjugate;
c. obtaining a sample of body fluid from the human or animal without dissociation of the target analyte from targeting moiety;
d. combining the sample of body fluid with a capture moiety capable of binding specifically to the analyte determinants of the targeting moiety: target analyte conjugate;
e. incubating the assay mixture to allow the immobilized capture moiety to bind specifically to either the target analyte or the labeled targeting moiety;
f. removing unbound targeting moiety and target analyte from the capture moiety;
g. detecting the bound conjugate on the capture moiety; and
h. determining the amount of the target analyte in the sample.
The body fluid sampled is generally saliva, blood or extracellular fluid. The targeting moiety used is preferably antibodies, soluble receptors, paratopic molecules, recombinant molecules with binding sites for the target analyte, or fragments thereof. The targeting moiety is preferably an antibody and most preferably a polyclonal antibody which recognizes many epitopes on the target analyte.
Generally, the targeting moiety is detectably labeled through the use of a labels which are preferably radioisotopes, affinity labels, enzymatic labels, or fluorescent labels. Preferably, the targeting moiety is labeled by linking the targeting moiety to a label which label can then be bound to a binding partner which is conjugated to an enzyme. More preferably, the label is a small molecule happen. Most preferably, the hapten is biotin.
The present invention also provides for reagent kits useful in performing the methods disclosed, providing:
(a) a first reagent containing a labeled targeting moiety specific for the target analyte and capable of forming a conjugate with the target analyte;
(b) a second reagent separated from said first reagent which contains a capture moiety for said conjugate; and
(c) a third reagent separated from said first and second reagents which contains a standard for the analyte.
Preferably, the targeting moiety is an antibody and the capture moiety is an antibody. More preferably, these antibodies are polyclonal. Also, it is preferred that the capture antibodies are immobilized on a solid support
The present invention also provides reagent kits useful in performing the disclosed methods, comprising: (a) a first container having paratopic molecules that immunoreact with a target analyte, and are operatively linked to an enzyme indicating means; (b) a second container having paratopic molecules that immunoreact with the target analyte at a site different from the first paratropic molecules but are not in the first container; and (c) one or more other containers comprising one or more of the following: a sample reservoir, a solid phase support, wash reagents, reagents capable of detecting presence of bound antibody from the second container, or reagents capable of amplifying the indication means.
Preferably, the paratopic molecules are detectably labeled through the use of a label selected from the group consisting of radioisotopes, affinity labels, enzymatic labels, and fluorescent labels. Most preferably, the paratopic molecules are detectably labeled through the use of fluorescent labeling agents are fluorochromes e.g., fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-1-naphthalenesulfony chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 or sulphonyl chloride (RB 200 SC).
In another embodiment, the present invention is directed to a method for monitoring immunological activity of a subject comprising measuring the concentration of a cytokine.
Accordingly, it is an object of the present invention to provide a method for accurately measuring the endogenous production of cytokines in humans or animals by sampling their blood.
Yet another object of the present invention is to provide a method by which the production of cytokines can be correlated to a pathological condition.
It is yet another object of the present invention to provide a method for evaluating and measuring cytokine production in response to behavioral perturbations.
It is a further object of the present invention to provide a method for evaluating cytokine production as a response to chemical, viral, parasite or bacterial challenges.
It is yet another object of the present invention to provide a method for ffi monitoring cytokine production during the course of an identified disease.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.